Vcx1p is a vacuolar Ca2+/H+ exchanger that transports Ca2+ into the vacuole with the energy provided by the H+ gradient in Saccharomyces cerevisiae. In the absence of Vcx1p activity, elevated levels of cytosolic free Ca2+ are prolonged, suggesting that Vcx1p plays a major role in cytosolic Ca2+ regulation. Activation of calcineurin, a phosphatase, results in the inhibition of Vcx1p. However, the molecular events downstream of calcineurin activation that result in Vcx1p inhibition are not known. This proposal presents the experiments by which I will: identify the residues of Vcx1 required for its regulation, determine whether Vcx1 activity is directly modulated by phosphorylation, identify the molecule(s) that interacts with Vcx1 and regulate its activity, and in-vitro reconstitute Vcx1 activity in order to kinetically characterize its regulation.